1. Field of the Invention
The present invention relates to an anti-human medullasin monoclonal antibody, process for producing the same and immunoassay using the same.
2. Description of the Related Art
Medullasin which is a kind of serine proteases occurs in granulocytes and the like, and is thought to widely play important roles in defense mechanism, including expression of inflammation, especially chronic inflammation. The amount of medullasin in granulocytes is increased in advanced stage of a number of chronic inflammatory diseases, and is normalized in remission stage. It is observed in patients suffering from multiple sclerosis that the amount of medullasin is prominently increased several days before the advanced stage and is normalized before remission. Multiple sclerosis is an intractable chronic inflammatory disease which mostly results in death in 10 to 15 years, which is characterized by localized demyelinated lesion in white matter of central nerve system and gliosis, and progresses repeating remission and aggravation. Although the cause of this disease has not been clarified, it is thought that this disease is a kind of autoimmune diseases in which autoantibodies attack the nerve tissue upon stimulation of the immune system by a virus or a bacterium. Its diagnosis is quite difficult and is now carried out by nuclear magnetic resonance imaging (MRI) or the like. However, MRI or the like requires a very large-scale equipment and high skill in the measuring operation, and is costly. Thus, a simple diagnosis method by which diagnosis of the disease, understanding of the state of the disease and assumption of the consequence can be accomplished is now being developed. For this, methods for measuring the activity of medullasin in granulocytes in the blood are now studied, and a method based on the ability of medullasin to inactivate apo-ornithine transaminase has been developed. However, since measurement of the activity of medullasin in granulocytes by an enzymatic chemical method is very complicated, simple immunoassay was tried and has been developed, which utilizes a polyclonal antibody.
However, the immunoassay for measuring human medullasin using a polyclonal antibody has drawbacks in that the reactivity of the antibody is not necessarily sufficient, the measuring time is long and the measuring operation is somewhat complicated. Thus, an immunoassay utilizing a monoclonal antibody, by which human medullasin can be measured quickly and simply, is demanded.
The present invention was accomplished under these circumstances and an object of the present invention is to provide a monoclonal antibody which specifically recognizes human medullasin and a method for specifically measuring human medullasin quickly and simply.
The present inventors intensively studied to attain the above-mentioned object and succeeded in obtaining an anti-human medullasin monoclonal antibody by culturing hybridomas prepared by cell fusion between antibody-producing cells recovered from an animal immunized with human medullasin separated and purified from human granulocytes and myeloma cells according to the method by Kxc3x6hler and Milstein, and discovered that human medullasin can be measured specifically, quickly and simply by using this antibody, thereby reaching the present invention.
According to the first aspect, the present invention provides an anti-human medullasin monoclonal antibody which specifically recognizes human medullasin.
According to the second aspect, the present invention provides a process for producing an anti-human medullasin monoclonal antibody comprising culturing hybridomas prepared by cell fusion between antibody-producing cells recovered from an animal immunized with human medullasin and myeloma cells, and recovering anti-human medullasin monoclonal antibody which specifically recognizes human medullasin from the culture in which the hybridomas are cultured.
According to the third aspect, the present invention provides an immunoassay comprising immobilizing human medullasin in a test sample by sandwiching the human medullasin between an anti-human medullasin monoclonal antibody immobilized on an insoluble carrier and a labelled anti-human medullasin monoclonal antibody by antigen-antibody reactions to form a complex, and quantifying the label in the complex.
By the present invention, an anti-human medullasin monoclonal antibody which specifically recognizes medullasin that is a kind of serine proteases existing in granulocytes was provided. By utilizing this monoclonal antibody, quick and simple immunoassay for measuring human medullasin was provided, which may be used for hemodiagnosis or the like for chronic inflammatory diseases, especially multiple sclerosis.